A proton and carbon 13 nuclear magnetic resonance study of neomycin B and its interactions with phosphatidylinositol 4,5-bisphosphate.

Abstract

The entire proton NMR spectrum of the aminoglycoside antibiotic neomycin B has been assigned at physiological pH by a combination of two-dimensional J-resolved and J-correlated and nuclear Overhauser enhancement difference spectroscopy. Unambiguous assignment of all four ring systems is possible without recourse to model or derivative compounds by observing nuclear Overhauser enhancements between as well as within rings. The subsequent assignment of the carbon 13 spectrum is simply achieved using two-dimensional heteronuclear J-correlated techniques. The proton NMR spectrum of a sonicated aqueous dispersion of the intracellular second messenger precursor phosphatidylinositol 4,5-bisphosphate is reported for the first time. The spectrum is consistent with a high degree of side chain unsaturation and a conformation for the myo-inositol head group, which appears highly mobile, in which all bulky substituents are equatorial (except the 2-hydroxyl). Addition of aliquots of phosphatidylinositol 4,5-bisphosphate to an aqueous buffered solution of neomycin B induces complex changes in the whole spectrum of the latter, including downfield shifts of differential magnitude for several well-resolved signals, viz. the anomerics, and the pair of methylene protons of the substituted cyclohexane. The complexation kinetics are fast on the NMR time scale at 25 degrees C. The binding results are discussed in terms of a tentative complexation geometry.